Several arenaviruses, chiefly Lassa (LASV) and Junin (JUNV) viruses, cause human hemorrhagic fever (HF) diseases that are associated with high morbidity and significant mortality, representing an important public health problem in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected clinically important human pathogen. Besides their public health burden, several arenaviruses, including LASV and JUNV are classified as NIAID Category A pathogens due to their potential as credible bioterrorism threats. Concerns posed by human pathogenic arenaviruses are further aggravated by the lack of FDA-approved vaccines and current anti- arenavirus therapy being limited to the off-label use of ribavirin, which is only partially effective and associated with significant side effects. The study of HF arenaviruses is limited by the requirement of BSL4 laboratories in order to manipulate live forms of the virus and also by secondary assays for virus detection. Development of valid single-cycle infectious virus surrogates would allow the study of HF arenavirus under less-strict BSL2 facilities, and viruses expressing a fluorescent reporter gene would facilitate the identification of prophylactic and therapeutic strategies using safe, sensitiv and specific assays that are compatible with High Throughput Screening (HTS) technologies. We have recently described, the generation of a single-cycle infectious, reporter-expressing, LCMV where we replaced the viral glycoprotein (GP) with a reporter green fluorescent protein (GFP), sciLCMV?GP/GFP. Infectious virus was achieved via genetic trans-complementation with BHK-21 cells constitutively expressing LCMV GP. This system allowed us to study multiple aspects of the virus and to develop screening assays to detect and quantify viral inhibitors as well as neutralizing antibodies. In this application, we propose to expand our technology to LASV by generating a single-cycle infectious, reporter- expressing, LASV (sciLASV?GP/GFP) that will allow the study of LASV under widely available and less restricted BSL2 facilities. This system will accelerate research on this important human pathogen, potentially identifying antivirals that target multiple steps in the replication cycle of LASV using HTS approaches. Moreover, we will also generate stable BHK-21 cell lines constitutively expressing GPs from representative strains of LASV lineages I-IV to produce GP-pseudotyped sciLASV?GP/GFPs. These pseudotyped sciLASV?GP/GFPs can be used to identify specific or broadly neutralizing antibodies against all LASV lineages, as well as to identify novel antiviral drugs targeting LASV GP-mediated cell entry. Overall, this proposal will overcome the major roadblock on LASV research that is currently imposed by the requirement of BSL4 laboratories.